Molluscan Research: Techniques for collecting, handling, preparing ...

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38 Simmons, J.E. (1991) Conservation problems of fluidpreserved specimens. In: Cato, P.S. & Jones, C. (eds.) Natural History Museums: Directions for Growth. Texas Tech University Press, Lubbock, pp 69–89. Smaldon, G. & Lee, E.W. (1979) A synopsis of methods for the narcotisation of marine invertebrates. Royal Scottish Museum, Information Series Natural History 6, 1–96. (Describes methods from literature, taxon by taxon. Numerous further references) Stamol, V. (1998) The main collection of Recent molluscs in the Croatian Natural History Museum. Natura Croatica 7, 159–167. Steedman, H.F. (Ed.) (1976) Zooplankton Fixation and Preservation. The UNESCO Press. 350 pp. Steinmann, W., Ebeling, R. & Goepel, U. (1975) Die Konservierung medizinischer und zoologischer Präparate in Phenoxetol. Der Präparator 21, 8–11. Steitz, E. & Stengel, G. (1984) Die Stämme und Klassen des Tierreichs. Verlag Chemie, Weinheim, Germany. Strasoldo, M. (1991) Anatomie und Ontogenie von Scissurella jucunda (Smith, 1890) und Anatomie von Anatoma sp. Dissertation University of Vienna, Austria, Vienna. 145 pp, 37 pls. Tennent, M. & Baird, T. (1985) The deterioration of Mollusca collections. Identification of shell efflorescence. Studies in Conservation 30, 75–83. Turner, S. (1998) The Book of Fine Paper. Thames & GEIGER ET AL. (2007) MOLLUSCAN RESEARCH, VOL. 27 Hudson, London. Travis, J. (2001) Alcohol on your breath needs not be that bad. Science News Sept. 22 nd . http:// www.sciencenews.org/ Tullberg, T. (1891) Über Konservierung von Evertebraten in ausgedehntem Zustand. Privately published, Stockholm. Ueshima, R. (2002) Simple methods for DNA preservation in molluscan specimens. Venus 61, 91–94. USI Chemicals. (1981) Ethyl alcohol handbook, 5 th Ed. National Distilleries and Chemical Corporation. Replaced by Equistar’s “Ethyl alcohol handbook”. Available at: http://www.lyondell.com/Lyondell/Products/ByMarket/ AdhesivesAndSealants/Solvents/Ethanol/ TechnicalInformation/index.htm [accessed 10/IV/2007] Wagstaffe, R. & Fidler, J.H. (1955) The Preservation of Natural History Specimens. Vol. 1. Invertebrates. H.F. & G. Witherby, London. Waller, R. & Strang, T.J.K. (1996) Physical chemical properties of preservative solutions. –1. Ethanol – water solutions. Collection Forum 12, 70–82. Warén, A. (1990). Ontogenetic changes in the trochoidean (Archaeogastropoda) radula, with some phylogenetic interpretations. Zoologica Scripta 19, 179–187. Zala, K., Pentcheff, N.D. & Wetzer, R. (2005) Laser-printed labels in wet collections: will they hold up? Collection Forum 19, 49–56.
TECHNIQUES FOR STUDYING SMALL MOLLUSCAN SPECIMENS 39 Appendix 1. Alphabetical list of chemicals used for work on micromolluscs. CAS numbers (Chemical Abstracts Service numbers, http://www.cas.org) are provided to facilitate further inquiries. List compiled by AW. Acetic acid, CAS no 64-19-7. Acetic acid is one of the oldest fixatives on record: in the eighteenth century vinegar (4–10% acetic acid content) was used to preserve hydras. It does not harden tissue; actually, it prevents some of the hardening that, without it, might be induced by subsequent alcohol treatment. In some techniques, however, acetic acid must be avoided because it dissolves certain cell inclusions, such as Golgi and mitochondria and calcareous material. Many lipids are miscible with acetic acid or are soluble in it. It neither fixes nor destroys carbohydrates. Acetic acid is used as a component of many fixatives, e.g., Bouin’s fluid. Its usefulness lies within its fixation of nucleoproteins, i.e., good nuclear fixation. Acetic acid (5–50%) can be used for decalcification. Acetone. CAS no 67-64-1. Water-free acetone causes strong shrinkage and is therefore not often used. It can be used for fixation of smears and unfixed sections. For histology, acetone is only used in combination with other fixatives, such as formalin and sublimate. Acetone is often used as a medium for critical point drying, but should be avoided because of its shrinking effect. Dry specimens directly from 100% ethanol, which is also soluble in carbon dioxide (drying medium). Acetone is useful for cleaning fat and remains of glue from shells , since it is a comparatively harmless chemical. Amylocaine hydrochloride (Stovaine). CAS no 532-59-2. Used for narcotisation. Slowly add 1% solution in water, drop by drop, to 100 ml of water with animals. For each drop give the chemical time to disperse and animals time to react. Benzamine compounds. Used for narcotisation. A 1–2% solution in water is slowly added, drop by drop, to 100 ml of water with animals. For each drop give the chemical time to disperse and the animals time to react. Bichromate. See chromic acid. Bleach. See sodium hypochlorite. Borax (disodium tetraborate). CAS no 1330-96-4. (Na 2 B 4 O 7 MW 201.2 and Na 2 B 4 O 7 , 10 H 2 O MW 381.4) is easily soluble in water, non toxic and its aqueous solutions are basic. Used for buffering formalin. The solubility of borax in 37% and 3.7% formalin is slightly more than 50 g /1000 ml, but varies ± 5 gram depending on the quality of the formalin (mainly due to concentration of methanol in the formalin). When buffering formalin, the variety with crystal water should be used because it dissolves much more easily. If too high concentrations of borax are used, it may recrystallise due to changed conditions in the solution, for example temperature or concentration of other substances. Such precipitations may be difficult to dissolve, but warm water usually helps. One gram per 10 litres of 37% formalin will raise the pH to 7.5–7.8 and is enough for several months; do not use more than 10 gram/litre. Dilution of 37% formalin buffered in this way to 3.7% will raise the pH about 0.9–1.0 units. For field work it may be useful to know that one teaspoon of borax contains 4.2 gram, one tablespoon, 11.5 gram. Concentrated solutions of borax interfere with carbon dioxide from the air and boric acid (H 3 BO 3 ) may precipitate, as milky clouds of very fine needles (easily visible at 12x) in the solution. This is a result of the equilibrium Na 2 B 4 O 7 + CO 2 + 6H 2 O 4H 3 BO 3 + 2Na + 2- + CO 3 . Such precipitations can be avoided by not adding more borax than necessary. Bouin’s Fluid. A common fixative used in histology. It is a mixture of 375 ml saturated aqueous picric acid, 125 ml stock formaldehyde (37% w/w), 25 ml glacial acetic acid. See under individual ingredients for hazards and safety precautions. Butyl acetate. CAS no 123-86-4. A good substitute for benzene and many other unpleasant non-polar organic solvents since it is less harmful. Hygienic limit for short term allowable air concentration is 150 ppm = 0.11 g/m 3 . It has a strong smell so you are certain not to stay anywhere with too much of it in the air. It can be used to dissolve various organic glues (not based on polyvinyl acetate) as well as for air-drying animals with some dermal skeleton, such as echinoderms and insects, with much less shrinkage. Transfer animals to butyl acetate via 95% and 100% ethanol. Even critical point dried specimens of soft-bodied animals can be cleaned or removed from a stub by dissolving the glue in butyl acetate, usually with little or no damage to the specimen, thanks to its low surface tension. Calcium carbonate. CAS no 471-34-1. Has a solubility constant of 3.36 x 10 -9 . This means that a saturated aqueous solution contains 5.8 mg/l, but due to formation of hydrocarbonate ions with aerial carbon dioxide, the solubility becomes higher with time. Calcium carbonate (as dolomite) is used for buffering commercial concentrated formalin, providing so-called neutral or histology quality. These attributes, however, cannot be stored for more than a couple of years because of the low solubility of dolomite. After that time the neutralizing capacity is exhausted. Calcium and magnesium salts are not good as buffers for specimens intended for histology because insoluble salts have a tendency to recrystallise in tissues (Quay 1974). Calcium phosphate. CAS no 7758-87-4. Has a solubility constant of 2.07 x 10 -33 and therefore sodiumphosphates are, not very good as buffers when fixing molluscs, since phosphate may replace carbonate in the shells. Carbowax. See Polyethylene glycol.
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